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Sporopollenin: A review of its chemistry,

palaeochemistry and geochemistry

J. Brooks & G. Shaw

To cite this article: J. Brooks & G. Shaw (1978) Sporopollenin: A review of its chemistry,
palaeochemistry and geochemistry, Grana, 17:2, 91-97, DOI: 10.1080/00173137809428858

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 Kina 17:91-97. 1978

;poropollenin: a review of its chemistry,

Balaeochemistry and geochemistry
BROOKS and G. SHAW

Brooks. J. 6; Shaw. B.: Sporopollenin: a review of its chemistry, palaeochemistry and

geochemistry. -GGnna 17: 91-97, 1978. Uppsala 29 September 1978. ISSN 0017-3134.
Sporopollenin. the tough resistant biopolymer that coats the outer walls of pollen grains,
spores and related microorganisms. considered "the most resistant organic material
known", accounts for the morphology and microstructure and for geological preservation
of palynomorphs. The stability of sporopollenin is thus the raison d'etrc of the science of
palynology. Although sporopollenin is such a unique and interesting material. very few
chemical studies had been made on it until research started at the University of Bradford in
the 1960's. In 1968. we discovered the relationship between the chemical nature and
properties of sporopollenin and those of oxidative polymers of carotenoids, and shed some
light on the chemical structure of sporopollenin. This review describes some of the
chemical, biochemical and geochemical studies on sporopollenin during the last ten years.

J . Brooks. Geoclieitiis(r3. Sectioii. Exploratioii Deportitietit, The British Ntitioiial Oil

Corporation. 150 St Viitceiit Street, Glasgoic G2 5 U . U~iitedKitigdoiii: G.S h n ~ cScliool
. of
C/ieitiisrty, Uiiil'ersitj of Brcit!/iortl,Brot!/iord. West Yorksliire. 8 0 7 IDP,Etigkcid.

Sporopollenins are probably the most resistant or-
ganic materials of direct biological origin found in
nature and in geological samples. They are the
chemical component that make up the outer wall of
pollen grains, spores and many related microor-
ganisms. The resistance of sporopollenins to
microbiological, chemical and physical degradation
permits study of the morphology and micro-
structure of such palynomorphs and is therefore
the basis of the science of palynology.
The earliest reported chemical studies o n the ex-
ine of pollen grains were by John (1814) and
Braconnot (1829). They both remarked upon the
resistance of the wall component t o chemical rea-
gents and introduced the term pollenin (pollenine)
to describe the .tough resistant chemical material
hich makes u p the outer wall of pollen grains. In
tcrms of modern usage, John and Braconnot used
thc word pollenin not only for the exine, but for the
spuroderm of spore coat including the underlying
;ntine of cellulose. Zetzsche (1928) introduced the
'.I #11 sporonin to describe the wall material from
spores of Lycopotlitrrn c l ~ t u t t t i / n and
, used the col-
lective name sporopollenin to describe the chemical
substance present in both pollen and spore walls
(Zetzsche et al. 1931).
Sporopollenin is a chemical term and is independ-
ent of botanical taxonomy. A recent and specific
definition for sporopollenin was proposed at the
International Symposium on Sporopollenin (Brooks
e t al. 1971) as : "sporopollenin is the name for the
highly resistant chemical substance o r substances in
the exine of the pollen wall and the exosporium of a
large number of spore walls" (Jonker 1971).
During the 1960s, a re-investigation of the
chemistry of sporopollenin was started a t the Uni-
versity of B n d f o r d using the many and varied mod-
em separation and analytical techniques. Com-
plimentary t o these chemical studies. we also
approached the problem of structural elucidation
from biochemical studies. Studies were carried out
on the biological synthesis of sporopollenin in the
anthers of various plants and showed that there was
a direct relationship between the synthesis of
92 J . Brooks arid G. S/imi*
sporopollenin and the production of carotenoids
(Brooks & Shaw 1%80. b, 1977).
In recent studies, radiochemical labelling tech-
niques have been used to help elucidate the struc-
ture of sporopollenin (see Green 1973). Techniques
have been developed in which “C labelled sodium
acetate, palmitate and mevalonic acid were injected
into young Lilirrni and Crrcirrbitn plants. The pollen
was collected and the carotenoids and sporopolle-
nin produced were found to be radio-labelled.
These radio-labelled carotenoids were re-intro-
duced into more plants (Lilirrrii and Crrcirrbitn) and
the sporopollenins collected were again found to be
labelled. More impressive and significant results
have been obtained from radio-labelling experi-
ments with the fungi, hlrrcor mucedo, Rhizoprrs
se.nrnlis and Nerrrospora crass0 zygospores. I4C
and ‘H labelled p-carotenes were successfully in-
corporated into the sporopollenin of the sexually
produced zygospores.
The ontogeny of pollen and spore walls has been
studied during the last decade (Heslop-Harrison
1%8, 1976, Rowley 1963, 1975, Godwin & Echlin
1968, Echlin 1971, Brooks & Shaw 19680, 1971.
1977). After release from the tetrad and during this
early development of the microspores, there is a
very rapid expansion of the spores and excretion of
new material on to the spore wall. During the stages
of growth of the microspores into mature or-
ganisms, various structural changes occur in the
cells of the tapetum of the anther. A distinctive
feature of these tapetum changes is the production
of “pro-orbicules” that are composed of sporopol-
lcnin precursors (“proto-sporopollenin”) suggesting
that the principal synthesis of sporopollenin may be
located in the tapetum.
Since sporopollenins are so resistant, pollen
grains and spores are deposited in sediments (pollen
analysis) and are often described as the most uni-
versal fossils. Interest in the thermal alteration of
spores (and other organic matter) has become of
interest to palynologists and geochemists for sev-
eral reasons:
( ( 1 ) there are correlations between sedimentary
properties and changes occurring in the chemical
structure of sporopollenins;
( b )the colour changes and chemical alterations of
thermally altered palynomorphs indicate the maxi-
mum temperature level to tvhich the sedimentary
organic matter has been heated. This in turn gives
valuable indications about the “thermal maturity”
of sedimentary organic matter and its potential for
production of hydrocarbons.
I. CHEMISTRY OF SOME LOWER
PLANT SPORES
It is well established that the cell wall constituents
of planktonic green algae (e.g. Clilorelln) show
significant similarity with the sporopollenin of vas-
cular plant spore exines (Atkinson et al. 1972,
Chaloner 1976). Resistant fossil “cysts”, equatable
with those of extant green algae, have been re-
corded back to the Devonian (Wall 1962) and
analysis of such fossil material gives a composi-
tion and properties similar to that of sporopolte-
nin (Brooks 1971). Similar, cyst-like bodies
(“Acritarchs”) have been well-documented in the
Lower Palaeozoic and Late and Early Precambrian
record (Downie 1973, Brooks et al. 1973, Brooks &
Shaw 1973). From these and many other records it
is clear that long before green plants effected land
migration they had already established biochemical
processes necessary to coat their spores with
this admirably resistant substance-sporopollenin
(Chaloner 1976).
Studies on the spores of two Lycopodiaceae
species Osmuricla regnlis and Selagitielli krciirssiu-
no, the fungal species Aspergihrs rriger. hlricor
niucedo. Nerrrospora crassn and Periicillirrrpi brcvi
and the algal materials Pediasfrirni r1iiple.r (var. re-
ticirlnta) and Cliarn cciralliiin all showed chemical
evidence for the presence of sporopollenin in their
walls. The Osriiitritln regalis spore walls were found
to contain almost 20 per cent of sporopollenin and
therefore, resembles two other species, Lyco-
podium clavatruii and Selagiiielln iirnrrssiaiia.
One of the more interesting of the lower plants
that we examined is hlrrcor nirrcedo and the forma-
tion of the (5)sexual zygospores in the fungus.
These zygospores contain about 4 per cent
sporopollenin, whilst the wall material of the asex-
ual spores is composed of polysaccharides.
Carotenoids are synthesised by both the (+) and
(-) strains of hlircor riirrcedu and the major compo-
nent is p-carotene. During zygospore formation in
Mrrcor there is an 100-fold increase in the produc-
tion of carotenoids and there is also a significant
increase in @carotene production in the mated
cultures of BlrrXesleti tricporci (Goodwin 1967).
Cliorinepliorrr crrcrrrbitti and 1’Iiyotiiyce.r
/hkc.slrcniirrr (Hocking 1965). The zygospores of
the fungus Rltizoj)rrs se.rrrtilis (considered related to
Mireor) do not produce cnrotenoids and do not con-
tain sporopollenin (Holleyhead 1972). Chemical and
histochemical studies on R1tizoprr.s scsrralis
zygospores showed that chitin and chitosan were
the major components of the wall.
I t . INCORPORATION 01' RADIO-
LABELLED CHEMICALS INTO
SPOKOPOLLENIN
A . Afrrcor rttrrceclo zygosj'ores
U-"C acetate, G - I T acetate, 2-"C acetate, 2-"C
mevalonate, G-"C 0-carotene and 15, 15'-3H
0-carotene have been incorporated into sporpolle-
nin material produced by the zygospores of rlfircor
rtrrrcch (Gooday et al. 1973).
In sporopollenins labelled with U-"C acetate,
'-IT acetate and 2 - ' T mevalonate and then
treated with 80 per cent ortho-phosphoric acid and
subscquent solvent extraction, showed no appreci-
able change in specific activity. This observation
sho\ved that the radio-labelled compounds were lo-
cated in the sporopollenin. An increased specific
x t i v i t y was observed in sporopollenin labelled with
IS, 1S'-311-carotene. This increase was accom-
panied by an equivalent loss in weight of the
sporopollenin material and suggested that residual
non-active polysacchandes were removed by
phosphoric acid treatment, showing that the label
tvas located in sporopollenin. Experiments showed
that /3-carotene and its precursors were incorpo-
rated into the ( t ) zygospore wall ofMtrcor itittccdo,
;ind established a useful technique for studying
\poropollenin using radio-labelled carotenoids.
11. Ncirrosporri crrtssci nscosporrs
15, 1S'-3111 0-carotene was incorporated into the wall
of the ascospores of Ncrrrosporci crussci. Incorpora-
tion of radio-labelled /+carotene (based upon
Nptcific activity recovered in the sporopollenin)
\\;IS 0.6 per cent and was significantly greater than
iii similar experiments with Mireor.
C. Circrirbitct j w j w polleri grciirts
U-"C :icetatc, 2J4C acetate and I-"C palmitate
ticre incorporated into sporopollenin component.
I):cr;idation and extraction methods on the wall
.IY\\ cd that the radio-labelled compounds were in-
.!liporated into and localised in the sporopollenin
!cti;il.
\',')
D. Lilirirtt ltenrji j d l c i i groiiis
2-"C mevalonic acid was incorporated into
antheraxanthin dipalmitate in the Liliirr~ anthers,
but "C-labelled antheraxanthin dipalmitate was not
significantly incorporated into the sporopollenin.
'this suggests that either the carotenoid ester was
not incorporated in its current state into the
sporopollenin, the labelled carotenoid ester was
present in the system in a form or at a time when it
was unable to take part in the exine biogenesis, or
the dilution factor within the plant tissue was too
great to allow significant identification of radio-
labelling in the sporopollenin material.
Green (1973) considered that the advanced state
of developnient of the Liliitrii Itciirji buds, at the
time of inoculation of the labelled materials, was an
important factor in the observed results.
111. SOME CHEhlICAL OBSERVATIONS
ON POLLEN WALL ONTOGENY
'the basic principles of pollen and spore wall
ontogeny have been reported in some detail by
Heslop-Harrison (1968, 1976). Rowley (1971, 1976),
Echlin (1971) and Dickinson (1971, 1976). Brooks
and Shaw ([%So, I), 1971, 19736) have recently
been able to explain and clarify many of these
biological observations on a chemical basis (Brooks
6r Shaw 1977). Extensions of these chemical expla-
nations from recent chemical and eytochemical
studies have also been made (Rowley and Brooks.
11 npubl.).
The first indications of patterning on pollen
grains is seen at the surface of young microspores
composing the tetrad. I t has bcen shown (Rowley
1976) that the specific characteristic of the
microspore "template" surface are determined by
glycocalyx (protein-polysaccharide complex)
macroniolecules associated with the plasma
membrane. This template consists of polyionic
molecules which form an agglutination bridge be-
tween closely spaced negatively-charged sites on
compounds within the exine, having a filamentous
or tape-like configuration. Filaments or lamellations
have been exposed from exines of mature
Lycopodiirrtt clciiwtiirti spores by oxidative degrada-
tion of the sporopollenin using ozone (Brooks 6r
Elsik 1974) and also by heat and pressure treatment
(Sengupta & Kowley 1974). These filamentous
stntctures have been isolated from the exines
of E'pild~iirtii ;ind Artmisin pollen using
Griiiiii 17
9-4 J . Brooks tiritl G . Sliritc.
2-aminoethanol and phenol extniction (Rowley
1976). A direct relationship exists between the
specific composition of the glycocalyx protrusions
morphology, exine morphology and similarities be-
tween the surfaces of the microspores and the
tapetal cell. The surface configuration of the de-
veloping microspores and that of the "pro-orbicule"
formed adjacent to tapetal cells is identical (Nilsson
& Robyns 1974).
When the microspores are released from the
tetrad, the morphology contained in the plasma
membrane-glycocalyx template is ready for deposi-
tion of sporopollenin. At approximately the same
time as the microspores are released, the tapctum
begins to produce sporopollenin precursors. The
main contributions of the tapetum chemicals ap-
pears to be in the deposition of the outer exine.
Concurrent with the production of these chemicals
in the tapetum. there is a progressive degeneration
of the secondary tapetum accompanied by produc-
tion of lipid material and of "pro-orbicules". These.
"pro-orbicules" consist of a strong solution of
carotenoids and are considered to be sporopollenin
precursors, consisting of "proto-sporopollenin"
(un- or partly polymerised material). This material
is transported across the anther cells to the surface
of the developing pollen wall surface. The poly-
merising "proto-sporopollenin" being deposited
onto the microspore wall surface into the "tem-
plate mould" composed on the glycocalyx fila-
ments (Brooks R: Shaw 1971, 1977).
Little is known about the relative rates of de-
velopment of exine deposition and formation in
these intermediate stages between release of the
microspore and the completion of the exine wall,
but from observations it appears that sporopollenin
accumulation occurs rapidly on to the exine.
IV. CHEIIICAL STRUCTUKE OF
SPOROPOLLENIN
A. C/icriiictil ci-idcrt ce
Oxidative degradation (using ozone) of natural
sporopollenins and of synthetic carotenoid poly-
mers shows excellent correlation between the prod-
ucts (Brooks & Shaw 1968a, h, 19736, 1977, and
unpublished results). Generally. the spectra of
compounds are very similar. showing sporopolle-
nins to have very similar chemical structures and to
be identical in structure to the synthetic carotenoid
polymers. Dctailcd examination of the spectra of
the di-carboxylic and mono-carboxylic acid oxida-
tive degradation products derived from sporopolle-
nins of different plant families show some minor.
but significant differences that may have potential
use in phytochemical correlations.
Pyrolysis-gas chromatography of sporopollenins,
synthetic carotenoid polymers and natural
carotenoids (Brooks & Shaw 1971, 19736, 1977.and
unpubl., Achari et al. 1973) gave almost identical
chromatograms showing that the sporopollenins de-
rived from angiosperm, gymnosperm and ferns and
lower plant spores have similar chemical structures
and also that they are similar to the synthetic
polymers.
A molecular structure for sporopollenin based
upon an oxidative polymer of cnrotenoids and
carotenoid esters has been reported (Brooks 6r
Shaw 1977, and unpubl.). This structure is sup-
ported by elemental analysis, chemical, biochemi-
cal, radiochemical and cytochemical evidence that
has been cam-ed out during the last few years.
X-ray diffraction studies on the natural and syn-
thetic polymers (Brooks, Joncs 6: Shaw, 1977
unpublished) confirm the chemical structure of
sporopollenin as an oxidative polymer of caro-
tenoids.
B . Cytocliciiiicnl cr*itlencc
Sporopollcnins are strongly osmiophilic (South-
worth 1973), showing the material to contain ali-
phatic unsaturated C-C double bonds. The polymer
absorbs U-V light (-290, 265 and 250 nm) (South-
worth 1969, Brooks & Shaw, unpubl.) and its
fluorescence (van Gijzel 1971) suggests the pres-
ence of conjugated C-C double bonds adjacent to
C-0 groups. Sporopollenin reacts with basic dyes
(nile blue A; azure L3 and toluidine blue) suggesting
the presence of weakly anionic groups such as
acidic-enolic compounds. Sporopollenins can be
distinguished from cutin and suberin by lack of stain
with Sudan IV (Southworth 1973) and from lignin
by its insolubility in dioxane (Ford 1971) and its
failure to stain with phloroglucinol (Heslop-
Harrison 1968).
These cytochemical observations clarify the
postulated chemical structure of sporopollenin. The
unsaturated C-C bonds provide the active sites for
staining with osmium tetroxide and would show
U-V and fluorescent spectra. The oxidation of the
carotenoid structiire leads to the formation of enolic
groups within long aliphatic carbon chains. In-

corporation of radio-labelled sodium acetate.
mevalonic acid and p-carotene compounds into de-
veloping pollen grains and spores during exine
synthesis shows conclusively that the sporopollenin
chemical structure is not related to lignin.
Sporopollenins are oxidative polymers of c a r e
tenoids and carotenoid esters.
V. GEOCHEMICAL STUDIES
OX SPOROPOLLENIN
Pollen grains and spores from different ranks of coal
a n d different sediments show gradual colour
changes from pale yellow, through light brown to
dark brown-black, due mainly to different thermal
histories. Considering experimental evidence, due
to difficulties in understanding all the various
p:irameters that may affect colour and chemical
changes in pollen and spores, it seems that such
changcs occur by coalification and carbonisation
processes (Brooks 19770). The processes depend
o n the different rates of heating, different times of
thermal alteration and the resulting chemical reac-
tions. Thermal alterations are considered to be the
most important parameter, but it is necessary to
note the effects of hydrostatic pressure gradients
;ind shear pressure gradients in regions subject to
deep burial (Gray & Boucot 1975).
Laboratory experiments have been used to study
the chemical changes in sporopollenins under
carbonisation processes (Gutjahr 1966, Correia
1971. Brooks 1970, 1971, 1977b, Combaz 1971,
Sengupta 1974). The pollen and spore walls show
colour and chemical changes that start at tempera-
tures between 100" and 200°C. Few studies have
been carried out on the progressive changes occur-
ring with sequential heating of pollen and spores,
but some results (Cook et al. 1972) suggest that
pollen and spores carbonise at different tempera-
tures, depending on their previous geothermal
history.
Pollen grains and spores that are heated for vary-
ing lengths of time at different temperatures show
very limited colour and chemical changes between
180'-200"C, but significant changes above 220°C
and above 170'C further significant changes occur
over shorter periods (Brooks 1971, 1977/>,
hlclntyre 1972. Sengupta 1974). Laboratory expen-
nicnts showed that when pollen and spore walls are
l!cated at lower temperatures (less than 180°C).
t..irt from the evolution of \vnter and small
Sporopollctriti 9S
amounts of methane and carbon dioxide, the
sporopollenin residue appears very little altered.
Sporopollenin heated to higher temperatures (great-
er than 220°C) produces more volatile and soluble
chemical products and shows significant alterations
in both colour and chemical changes in a relatively
short time (Brooks 1977b).
Pollen and spores show changes in colour and
chemical structure with temperature at different
rates than dinoflagellates and acritarchs (Correia
1971) and they show earlier changes than
chitinozoa.
Pollen grains and spores are the most sensitive
indicators of thermal changes in any particular re-
gion of a sediment, especially in areas of relative
low thermal activities and gradients. These facts
have resulted in palynomorphs-particularly pollen
and spores-being used in geothermal studies on
sediments. Although these techniques have wide
applications (see Brooks & Niklas 1977). their ma-
jor current use is in petroleum exploration. Using
relationships between pollen grain and spore colour,
translucency and fluorescence, chemical changes,
electron spin resonance (ESR) and thernial
gravimetnc analysis (TGA) and geological condi-
tions, it is now possible for organic geochemists and
palynologists to determine the regional thermal
history of sediments and their potential for
hydrocarbon generation.
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Atkinson, A. W.. Gunning, B. E. S . & John. P. C. L. 1972.
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